Significance of CpG Methylation in Cancer
CpG islands are short genomic segments in the promoter regions of most human genes that are enriched in the dinucleotide CpG. CpG islands are unmethylated in normal tissues but become progressively more methylated in cancer cells, leading to repression of genes that control the cell cycle.
Cancer is actively avoided through the expression of numerous tumor-suppressor genes that regulate the cell division cycle and mediate interactions among cells. Studies of benign and malignant tumors have shown that cancer develops in a multi-step process where randomly accumulated changes either enhance the expression of proto-oncogenes or reduce the expression or function of tumor-suppressor and DNA repair genes (Nowell et al. (1976) Science 194:23-28). Somatic mutations account for some of these changes in tumor-suppressor and DNA repair genes. However, it has recently become apparent that epigenetic changes such as DNA hypermethylation and hypomethylation also play a large role in the development of cancer through inactivation of tumor suppressors or enhancement of proto-oncogenes (Esteller (2007) Nat. Rev. Genet. 8:286-98; Feinberg & Vogelstein (1983) Nature 301:89-92; Paluszczak & Baer-Dubowska (2006) J. Appl. Genet. 47:365-75; Vogelstein & Kinzler (1993) Trends Genet. 9:138-41). Virtually all systems for avoiding transformation can be disabled by epigenetic inactivation via hypermethylation (Esteller et al. (2001) Cancer Res. 61:3225-29). These changes occur at discrete clusters of CpG sites but in the context of a global reduction in cytosine methylation (Feinberg & Vogelstein (1983) Nature 301:89-92; Gama-Sosa et al. (1983) 11:6883-94). The pathways for detecting and dealing with DNA damage/replication errors can also be disabled by methylation changes, allowing additional mutations to accumulate and cause genomic instability. The expression of key genes can be altered either by an increase (hypermethylation) or a decrease (hypomethylation) of methylation at promoter CpG islands. Hypermethylation at promoter CpG islands of tumor suppressor genes results in a decrease in their expression level.
Hypermethylation of CpG promoter islands occurs at an early stage of cancer development and is found in virtually all tumors, making it potentially very useful as a diagnostic marker (reviewed in Esteller (2007) Nat. Rev. Genet. 8:286-98; Paluszczak & Baer-Dubowska (2006) J. Appl. Genet. 47:365-75), allowing cancer to be noninvasively detected in the early stages when treatment is most effective. Hypermethylation of the promoter region of genes such as DAP kinase, p16, and MGMT can be detected in the sputum of smokers up to 3 years prior to the diagnosis of squamous cell lung carcinoma (Belinsky et al. (2006) Cancer Res. 66:3338-44; Palmisano et al. (2000) Cancer Res. 60:5954-58). Similarly, hypermethylation of a small panel of genes may be a valuable early detection method for non-small cell lung cancer (Kim et al. (2004) J. Clin. Oncol. 22:2363-70). Hypermethylation of a small panel of genes was detected in the early stages of breast cancer from nipple aspirate fluid but was not detected in normal or benign breast tissue (Krassenstein et al. (2004) Clin. Cancer. Res. 10:28-32). Hypermethylation of GSTP1 is found in >90% of prostate cancers, can be detected via analysis of urine, and is not found in normal tissue or benign prostate lesions (Cairns et al. (2001) Clin. Cancer Res. 7:2727-30; Henrique & Jeronimo (2004) Eur. Urol. 46:660-69: 16 Jeronimo et al. (2004) Clin. Cancer Res. 10:8472-78). Hypermethylation of DAP kinase and p16 are found in premalignant cells in bladder cancer and the early stages of gastric carcinoma (Kang et al. (2001) Cancer Res. 61:2847-51; Tada et al. (2002) Cancer Res. 62:4048-53). A recent genome-wide screen of promoter methylation patterns in various cancers suggested that primary lung, breast, colon, and prostate cancers may share a promoter hypermethylation signature that can be used for early detection screening (Shames et al. (2006) PLoS Med. 3:e486).
In addition to acting as markers for early cancer detection, hypermethylation of promoter CpG islands may act as markers of tumor prognosis and potential for relapse. Hypermethylation of E-cadherin is associated with shorter disease-free survival in gastric and tongue cancer (Chang et al. (2002) Cancer 94:386-92; Graziano et al. (2004) Clin. Cancer Res. 10:2784-89). Hypermethylation of DAP kinase, p16, and EMP3 are linked to tumor aggressiveness in lung, colorectal, and brain cancer (Esteller (2005) Ann. Rev. Pharmacol. Toxicol. 45:629-56). Hypermethylation of calcitonin in lymphoblastic leukemia is associated with an increased risk of relapse. DNA hypermethylation profiles could potentially be used to determine cancer subtype, which can help determine risk and prognosis. One recent study found a clustering of neuroblastomas according to risk based on the methylation profile of a panel of genes (Alaminos et al. (2004) J.N.C.I. 96:1208-19). Another recent study found that subtypes of renal cancer could be distinguished based on multiple gene methylation profiles (Gonzalgo et al. (2004) Clin. Cancer Res. 10:7276-83). Finally, methylation profiling has been used to determine a multifocal versus metastatic origin for multiple hepatocellular carcinomas, which may lead to differential treatment and prognosis (Anzola et al. (2004) Scand. J. Gastroenterol. 39:246-51).
Hypermethylation events can also be used as a predictor of chemotherapy response and as a tool for monitoring the success of chemotherapy and potential for relapse. Knowledge of the methylation level of certain genes in primary tumors may indicate tumor response to various chemotherapeutic agents, allowing therapy to be tailored to the individual. This would increase the efficiency of treatment and spare patients unnecessary side effects of drugs with a low probability of shrinking tumor size. Hypermethylation of DNA repair genes can confer chemosensitivity, while hypermethylation of proapoptotic genes can confer chemotherapy resistance (Esteller (2000) Eur. J. Cancer 36:2294-2300; Soengas et al. (2001) Nature 409:207-11). Methylation of ATM correlates with increased radiosensitivity of colorectal tumor cells (Kim et al. (2002) Oncogene 21:3864-71). Sensitivity of gastric cancers to microtubule inhibitors depends on the hypermethylation state of CHFR (Satoh et al. (2003) Cancer Res. 63:8606-13). MGMT hypermethylation can predict the response of gliomas to the chemotherapeutic drugs BCNU and temozolomide, of melanomas to fotemustine, and of tumors to cyclophosphamide (Esteller (2000) Eur. J. Cancer 36:2294-2300; Christmann et al. (2001) Int. J. Cancer 92:123-29; Esteller et al. (2002) J. Natl. Cancer Inst. 94:26-32; Hegi et al. (2005) N. Engl. J. Med. 352:997-1003). Hypermethylation of COX2 can reduce the effectiveness of COX2 inhibitors (Toyota et al. (2000) Cancer Res. 60:4044-48). The hypermethylation states of phosphoserine aminotransferase or ESR1 are the best predictors of breast cancer response to tamoxifen therapy (Martens et al. (2005) Cancer Res. 65:4101-17. Widschwendter et al. (2004) Cancer Res. 64:3807-13), and the hypermethylation state of RASSF1A can be used to monitor the efficiency of the therapy (Fiegl et al. (2005) Cancer Res. 65:1141-45). Finally, hypermethylation of a panel of genes can be used to detect and monitor residual disease or relapse in natural killer cell lymphoma, and has a higher sensitivity than histological tests (Siu et al. (2003) Br. J. Haematol. 122:70-77).
Methods for Detecting Methylated-CpGs
A number of methods have been used to detect methylated-CpG (mCpG) in target DNA. The three primary methods in current use are detailed below.
Bisulfite Methods. The most commonly used methylation detection methods utilize bisulfite modification of DNA, resulting in deamination of cytosine residues to uracil while leaving the methylated cytosines unchanged. Upon PCR amplification, the methylated cytosine is copied to cytosine and uracil is copied to thymine. As a result, the retention of cytosine at a specific position indicates methylation. The modified DNA can then be analyzed by sequence analysis, methylation-specific PCR (MSP) (Herman et al. (1996) Proc. Natl. Acad. Sci. USA 93:9821-26), hybridization (e.g. to a microarray or blot) and the like. In MSP, a pair of methylation-specific oligonucleotide primers is added to the bisulfite-treated DNA and PCR is performed in order to amplify the target DNA. Fluorescence-based quantitative real-time PCR can also be performed on bisulfite-modified DNA (Eads et al. (2000) Nucl. Acids Res. 28:E32; Zeschnigk et al. (2004) Nucl. Acids Res. 32:e125).
Commercial kits, reagents and systems employing bisulfite treatment for analyzing mCpG are available. Epigenomics (Berlin) has two variants of the MethylLight assay, adaptations of quantitative real-time PCR, called Quantitative MethylLight (QM) and Heavy Methyl (HM). QM utilizes Taqman® probes to generate the fluorescent signal. During the course of amplification, the fluor is cleaved from the Taqman® probe resulting in fluorescence that can be detected in real-time (Wojdacz & Dobrovic (2007) Nucl. Acids Res. 35:e41). HM is an adaptation of QM in which blocker oligonucleotides are added to the reaction. These blocker oligonucleotides prevent the amplification from unmethylated DNA, resulting in increased assay sensitivity (Cottrell et al. (2004) Nucl. Acids Res. 32:e10). Pyrosequencing® is also utilized for methylation quantitation from bisulfite-modified DNA, as exemplified by the Pyro Q-CpG™ system from Biotage (Uppsala, Sweden; Tost et al. (2003) Biotechniques 35:152-56).
Although bisulfite modification is a widely used and an established procedure for detecting CpG methylation, bisulfite treatment can destroy a large percentage of the input DNA, resulting in limited sensitivity and requiring large quantities of sample DNA. Extensive degradation can introduce sampling errors when few molecules are long enough to be amplified (Ehrich et al. (2007) Nucl. Acids Res. 35:e29). Furthermore, the assays are time-consuming, require a harsh base denaturation step, and have a high-probability of false-positive results due to the incomplete cytosine deamination during bisulfite treatment.
Methylation-Sensitive Restriction Enzyme Digestion Methods. A second method for detection of mCpG in DNA relies on restriction enzyme analysis. DNA is treated with either a MSRE (methylation-sensitive restriction enzyme) or a MDRE (methylation dependent restriction enzyme), amplified and then analyzed by microarray or gel electrophoresis. MSREs such as HpaII and AciI cut a sequence only if it is unmethylated. MDREs are restriction enzymes that require methylation of a DNA sequence for cleavage. By treating a sample of DNA with either of these enzymes and subsequent comparison to a control sample, the methylation state of a DNA sample can be determined. If digestion of a specific DNA sample occurs after treatment with a MDRE, then the DNA is assumed to be methylated. Conversely, if the DNA is uncut when treated with a MSRE, then this sample is also assumed to be methylated. By comparing the amount of cut vs. uncut DNA, the level of methylation can be estimated. A common read-out for this type of methylation analysis is the subsequent amplification and fluorescent labeling of the digested DNA. The fragments can then by hybridized to a library microarray and analyzed or simply resolved by electrophoresis.
Commercially available systems include Orion's MethylScope®, a system that utilizes restriction enzymes and a microarray read-out (Lippman et al. (2004) Nature 430:471-76), and MethyScreen, which employs quantitative real-time PCR (Ordway et al. (2006) Carcinogenesis 27:2409-23).
An advantage of MSRE/MDRE digestion is that no pre-treatment of the DNA is necessary, although it is often performed in conjunction with bisulfite treatment of DNA in a procedure called COBRA (Xiong & Laird (1997) Nucl. Acids Res. 25:2532-34). Some disadvantages with this application are that it is a rather lengthy procedure and is dependent on the presence of recognition sequences. Furthermore, this approach is relatively inefficient, which can reduce the reliability of the results.
Chromatin Immunoprecitipation Methods. A third method that is commonly employed for detecting mCpG is chromatin immunoprecipitation (ChIP). Typically, cells are fixed, and then methylated DNA is immunoprecipitated by the use of antibodies specific for methyl binding proteins. The resulting DNA is amplified, labeled and analyzed by hybridization in a microarray assay. The advantages of this method are that the assay can be performed from live cells with little or no DNA purification required. The assay also has increased sensitivity, as unwanted and contaminant DNA are removed prior to analysis. However, the procedure is very time-consuming, involves several steps and requires expensive reagents. Some assays may take as long as five days to complete.
Given the importance of CpG methylation in cancer development and progression, a rapid, reliable, and sensitive test for methylated CpG DNA would provide an important and useful tool for cancer detection, diagnosis and monitoring.